The long term goals of this proposal are to understand the functions of the transcription factor TFIID and to identify and analyze factors with which it interacts. TFIID binds to the TATA regions of eukaryotic promoters and is required for transcription initiation by RNA polymerase II in vivo and in vitro. Mutations in the yeast SPT15 gene, which encodes TFIID, were isolated as suppressors of insertion mutations and cause a variety of mutant phenotypes and transcriptional defects. From a set of genetically characterized spt15 mutants, the sptl5 mutations will be sequenced and the mutant TFIID products will be studied biochemically. In addition, other classes of sptl5 mutations will be made in vitro and isolated in vivo. Analysis of different classes of spt15 mutants will correlate in vivo phenotypes, in vitro defects, and amino acid changes in TFIID. Analysis of spt15 mutants will also address the roles of TFIID in regulation of gene expression. To identify other components important for transcription initiation in vivo, unlinked suppressors of sptl5 mutations will be studied. One suppressor already isolated indicates that the SPT3 gene product physically interacts with TFIID. This possibility will be tested by biochemical and genetic experiments. In addition, other suppressors of sptl5 mutations will be isolated. The genes identified by these screens will be characterized to begin to elucidate their interactions with TFIID and their roles in transcription initiation. Given the conservation of transcription initiation in eukaryotes, an understanding of these functions will be relevant to understanding this process in humans.